lipoglycans from glycans by hydrophobic interaction chromatography, and (3) size fractionation of lipoglycans by size-exclusion
chromatography [57].
1. Dry delipidated cells obtained in Subheading 3.1.
2. Resuspend them in a minimum amount of deionized water.
3. Disrupt them by probe sonication in ice.
4. Reflux the cell lysate in a large volume of 50% ethanol in
deionized water for 4 h at 65
C.
5. Recover the supernatant by centrifugation at 3000 Â g.
6. Repeat steps 4 and 5 until the supernatant is colorless.
7. Combine the ethanol/water extracts and evaporate to dryness
under vacuum with a rotary evaporator (see Note 3).
8. Suspend the dried ethanol/water extract in PBS at a concentration of 0.5 mg/mL.
9. Add an equal volume of phenol saturated with PBS and shake
with a magnetic stirrer in a screw-cap tube or flask for 2 h at
80
C.
10. Allow the sample to cooldown at room temperature and separate the aqueous and phenol layers by centrifugation at room
temperature at 3000 Â g.
Table 3
Off-diagonal spin system identified on 2D1 H-H-COSY-NMR spectra
Structural
function
Spin system (δ, ppm)
Mycolic unit
pattern
– 3.65 (–CH–OH), 2.44 (CH wearing the ramification), 1.4 (αCH 2 -CHOH–)
– 2.44 (CH wearing the ramification), 3.65 (–CH–OH), 1.69 and 1.57
(nonequivalent protons of the CH 2 of the ramified chain)–
Keto pattern
– 2.49 (–CH 2 –CHCH 3 –CO–), 1.29 (–CH 2 –CHCH 3 –CO–), 1.05
(d, –CH 2 –CHCH 3 –CO–)
– 2.35 (–CO–CH 2 –CH 2 –), 1.54 (–CO–CH2–CH2–)
Cyclopropane
pattern
Cis: 0.64, 0.56, À0.34
Trans: 0.44, 0.20–0.08
Insaturation
pattern cis
– 5.35(–CH 2 –CH 2 –CH¼CH–CH 2 –CH 2 –), 2.05
(–CH 2 –CH 2 –CH¼CH–CH 2 –CH 2 –), 1.35
(–CH 2 –CH 2 –CH¼CH–CH 2 –CH 2 –)
Methoxy pattern – 2.95 (–CHCH 3 –CHOCH 3 –CH 2 –), 1.60 (–CHCH 3 –CHOCH 3 –CH 2 –), 1.39
(–CHCH 3 –CHOCH 3 –CH 2 –)
– 3.35 (–CHCH 3 –CHOCH 3 –CH 2 –)
– 1.60 (–CHCH 3 –CHOCH 3 –), 0.84 (–CHCH 3 –CHOCH 3 –)
Epoxy pattern
– 2.71 (–CH 2 –CHOCH–CH 2 –), 1.50 –CH 2 –CHOCH–CH 2 –
138
Marie-Antoinette Lane ´ elle et al.
chromatography [57].
1. Dry delipidated cells obtained in Subheading 3.1.
2. Resuspend them in a minimum amount of deionized water.
3. Disrupt them by probe sonication in ice.
4. Reflux the cell lysate in a large volume of 50% ethanol in
deionized water for 4 h at 65
C.
5. Recover the supernatant by centrifugation at 3000 Â g.
6. Repeat steps 4 and 5 until the supernatant is colorless.
7. Combine the ethanol/water extracts and evaporate to dryness
under vacuum with a rotary evaporator (see Note 3).
8. Suspend the dried ethanol/water extract in PBS at a concentration of 0.5 mg/mL.
9. Add an equal volume of phenol saturated with PBS and shake
with a magnetic stirrer in a screw-cap tube or flask for 2 h at
80
C.
10. Allow the sample to cooldown at room temperature and separate the aqueous and phenol layers by centrifugation at room
temperature at 3000 Â g.
Table 3
Off-diagonal spin system identified on 2D1 H-H-COSY-NMR spectra
Structural
function
Spin system (δ, ppm)
Mycolic unit
pattern
– 3.65 (–CH–OH), 2.44 (CH wearing the ramification), 1.4 (αCH 2 -CHOH–)
– 2.44 (CH wearing the ramification), 3.65 (–CH–OH), 1.69 and 1.57
(nonequivalent protons of the CH 2 of the ramified chain)–
Keto pattern
– 2.49 (–CH 2 –CHCH 3 –CO–), 1.29 (–CH 2 –CHCH 3 –CO–), 1.05
(d, –CH 2 –CHCH 3 –CO–)
– 2.35 (–CO–CH 2 –CH 2 –), 1.54 (–CO–CH2–CH2–)
Cyclopropane
pattern
Cis: 0.64, 0.56, À0.34
Trans: 0.44, 0.20–0.08
Insaturation
pattern cis
– 5.35(–CH 2 –CH 2 –CH¼CH–CH 2 –CH 2 –), 2.05
(–CH 2 –CH 2 –CH¼CH–CH 2 –CH 2 –), 1.35
(–CH 2 –CH 2 –CH¼CH–CH 2 –CH 2 –)
Methoxy pattern – 2.95 (–CHCH 3 –CHOCH 3 –CH 2 –), 1.60 (–CHCH 3 –CHOCH 3 –CH 2 –), 1.39
(–CHCH 3 –CHOCH 3 –CH 2 –)
– 3.35 (–CHCH 3 –CHOCH 3 –CH 2 –)
– 1.60 (–CHCH 3 –CHOCH 3 –), 0.84 (–CHCH 3 –CHOCH 3 –)
Epoxy pattern
– 2.71 (–CH 2 –CHOCH–CH 2 –), 1.50 –CH 2 –CHOCH–CH 2 –
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